C-reactive protein (CRP) is an acute-phase protein synthesized by the liver, widely accepted as a biomarker for cardiovascular disease and inflammation (May and Wang 2007, Miller et al. 2007, Mygind et al. 2011, Pai et al. 2008). Generally, levels in plasma are less than 2.0 mg/L for healthy individuals (Vikholm-Lundin and Albers 2006), but increase up to 1000 fold during an acute phase of inflammation (Gabay and Kushner 1999). The American Heart Association and the United States Centre for Disease Control have suggested three categories of CRP concentration for the evaluation of cardiovascular disease risk: a CRP concentration below 1.0 mg/L representing low risk, a 1.0 to 3.0 mg/L range average risk, and levels above 3.0 mg/L representing high risk (Kushner and Sehgal 2002, Lee et al. 2011). The reliable and early quantification of this target if often, then, cited as a means of improving the outcome of cardiovascular or inflammatory disease through appropriate intervention or treatment.
Currently, a number of CRP testing methods are available in clinical laboratories using turbidimetric and nephelometric technologies (Roberts et al. 2000, Roberts et al. 2001), or human CRP enzyme-linked immunosorbent assay (ELISA) kits. However, these methods are generally not suitable for the clinical practice as they are either not sensitive enough, time-consuming, prone to false negatives or cost-ineffective (Pearson et al. 2004). CRP quantification methods based on surface plasmon resonance (SPR) (Hu et al. 2006, Meyer et al. 2006), piezoelectric microcantilevers (Wee et al. 2005), quartz crystal microbalance technology (Kim et al. 2009), microfluidics (Lee et al. 2011) and electrochemistry (Buch and Rishpon 2008, Centi et al. 2009), have been developed during the past few years. Among these, electrochemical assays promise most in terms of low cost, flexibility and sensitivity. Electrochemical impedance spectroscopy (EIS), in particular, can sensitively monitor the changes in capacitance or charge-transfer resistance associated with material binding at specifically prepared receptive electrode surfaces and requires no prior labelling (Bogomolova et al. 2009, Rodriguez et al. 2005). In recent years a number of CRP assays by EIS have been reported. To date, however, these have been either of limited sensitivity (Vermeeren et al. 2011), not demonstrably specific (Hennessey et al. 2009), or to not encompass a clinically relevant range (Chen et al. 2008, Qureshi et al. 2010).
This subject-matter of this application relates to the development of a robust and highly sensitive assay for CRP in whole and dilute blood serum across the entire clinically relevant range. The technique can also be applied to other markers. The interfaces are readily prepared, exhibit very good selectivity and are re-useable after assay with no apparent loss of sensitivity. We have, additionally, considered the importance of receptive layer initial resistance in subsequently observed sensitivity and demonstrated not only a clear correlation but also an ability to tune, and therefore optimise, receptive film characteristics.